Source diversity of Artemia enrichment boosts goldfish (Carassius auratus) performance, β-carotene content, pigmentation, immune-physiological and transcriptomic responses

This study aimed to assess the impact of spirulina and/or canthaxanthin-enriched Artemia on the goldfish (Carassius auratus) growth, pigmentation, blood analysis, immunity, intestine and liver histomorphology, and expression of somatolactin (SL) and growth hormone (GH) genes. Artemia was enriched with spirulina and/or canthaxanthin for 24 h. Goldfish (N = 225, 1.10 ± 0.02 g) were tested in five experimental treatments, three replicates each: (T1) fish fed a commercial diet; (T2) fish fed a commercial diet and un-enriched Artemia (UEA); (T3) fish fed a commercial diet and spirulina-enriched Artemia (SEA); (T4) fish fed a commercial diet and canthaxanthin-enriched Artemia (CEA); and (T5) fish fed a commercial diet and spirulina and canthaxanthin-enriched Artemia (SCA) for 90 days. The results showed that the use of spirulina and/or canthaxanthin increased performance, β-carotene content and polyunsaturated fatty acids of Artemia. For goldfish, T5 showed the highest growth performance, β-carotene concentration and the lowest chromatic deformity. T5 also showed improved hematology profile, serum biochemical, and immunological parameters. Histomorphology of the intestine revealed an increase in villi length and goblet cells number in the anterior and middle intestine, with normal liver structure in T5. SL and GH gene expression in the liver and brain differed significantly among treatments with a significant increase in enriched Artemia treatments compared to T1 and T2. In conclusion, the use of spirulina and/or canthaxanthin improved performance of Artemia. Feeding goldfish spirulina and/or canthaxanthin-enriched Artemia improved performance, β-carotene content, pigmentation, health status and immune-physiological response.


Preparation of un-enriched and enriched Artemia nauplii
The brine shrimp (Artemia salina) eggs were purchased commercially from a brine shrimp (El-Max research station, Alexandria, Egypt).Artemia cysts weighing 0.004 g (250,000 cysts g −1 ) were incubated in 1 L of salted water (30-33 ppt) in a plastic bottle with a conical bottom that was mildly aerated (constant oxygen supply) and illuminated with 2000 lx during the hatching process.The water used for hatching and enriching Artemia had a pH of 8.13, total dissolved salts (TDS) of 4.22 g L −1 , and a temperature of 28 °C42 and Artemia hatched 24 h later.Cyst shells float to the surface at the same time.For 24 h, a large portion of Artemia was enriched with three carotenoid sources: spirulina (0.5 g L −1 ) (SEA), canthaxanthin (0.5 g L −1 ) (CEA), and a combination of both spirulina and canthaxanthin (0.5 and 0.5 g L −1 respectively) (SCA) representing T3, T4, and T5 respectively, whereas T2 was fed un-enriched Artemia (UEA) and T1 was fed a commercial diet only according to 43 .Spirulina and canthaxanthin were dissolved in the water and mixed thoroughly with the water in the plastic bottle with a conical bottom.To collect enriched Artemia, the aeration was removed to allow the nauplii to gather at the conical portion of the plastic bottle.Enriched Artemia was sucked from the plastic bottle after five minutes, collected on fine gauze, and washed with distilled water to remove any adherent food particles.Artemia was dried and weighed on a filter paper.Enriched Artemia was produced on a daily basis in order to provide goldfish with enriched Artemia on a constant basis.

Goldfish and rearing systems
The goldfish fry (N = 400, 1.10 ± 0.02 g) were obtained from a private commercial hatchery and transported in polyethylene plastic bags (1 water:2 pure oxygen) to the glass aquarium laboratory.Tap water was used in this experiment and aerated for 72 h to ensure that there is no residual chlorine content.Water temperature was 28.45 °C, pH was 7.20, dissolved oxygen was > 6 mg L −1 , and unionized ammonia was less than 0.001 mg L −1 .The water was filtered through a 0.45 μm membrane filter prior to use.The goldfish were acclimated for two weeks before the start of the trial.The proximate composition of the commercial diet used during the adaptation period contained 90% dry matter (DM), 44% crude protein, 9% ether extract, 7% ash, 36.62%Nitrogen-free extract (NFE), 3.38% crude fiber and 4520 kcal kg −1 gross energy.After the acclimatization period, the fish (n = 225) were distributed into 15 glass aquariums (45 cm × 30 cm × 25 cm and 30 L capacity) representing five experimental treatments and three replicate each (15 fish per aquarium).Each aquarium was supported with an aeration system from a radial compressor with fine-porous stones placed at the bottom of each aquarium.The experimental units were placed in a wet laboratory with artificial lighting and a controlled photoperiod (12 light:12 dark) and the experiment lasted for 90 days.

Experimental treatments
The experimental fish were randomly allocated into five treatments as follows: treatment one (T1): goldfish fed a commercial diet (10% of body mass, adjusted according to fish till reach 5% at the end of 4th week then continue with 5% till the end of 90 days experimental period); treatment two (T2): fish fed commercial diet and un-enriched Artemia at 200 pieces per goldfish/day [44][45][46] ; treatment three (T3): fish fed commercial diet and spirulina-enriched Artemia at 200 pieces per goldfish/day; treatment four (T4): fish fed commercial diet and canthaxanthin-enriched Artemia at 200 pieces per goldfish/day; and treatment five (T5): fish fed commercial diet and Artemia enriched with both spirulina and canthaxanthin at 200 pieces per goldfish/day.The goldfish received three equal meals (9 a.m., 12 p.m., and 3 p.m.) per day containing commercial diet for T1 only.For T2, T3, T4, and T5, fish received one meal (at 9 a.m.) containing a commercial diet of the same amount introduced to fish in T1 and two Artemia meals (at 12 p.m., and 3 p.m.).The success of the enriched process is illustrated in (Fig. 1).Images of un-enriched and enriched Artemia with spirulina, canthaxanthin, or both were taken with a Leica microscope (LEICA ICC50W) (www.leica-micro syste ms.com).The approximate composition of the commercial diet contained 90% dry matter (DM), 44% crude protein, 9% ether extract, 7% ash, 36.62%Nitrogen-free extract (NFE), 3.38% crude fiber and 4520 kcal kg −1 gross energy.Unconsumed feed was collected daily, and feces were removed and 25% of the water was refreshed every 2 days.www.nature.com/scientificreports/

Experimental measurments
Artemia Proximate composition and characteristics of Artemia.Proximate composition analysis of moisture, crude protein, crude lipid, fiber, and ash of experimental finished diets were determined according to analytical procedures described in 47 .For each analysis, triplicate samples were used.Every day, the survival rate of each treatment was calculated by observing and calculating the number of Artemia.A high-precision microscope (Nikon, Japan) was used to measure the length of each Artemia.A 100 mL water sample was taken from each replicate tank using the random sampling technique to determine the density and biomass of Artemia.The biomass of Artemia was measured on a dry weight basis with a microbalance (Electrical Analytical Balance, OSK 11325A), and the size of Artemia was measured isobar on a LEICA ICC50 (www.leica-micro syste ms.com).
Total carotenoid assessment of Artemia using UV-VIS spectrophotometer.Total carotenoids content was determined using a spectrophotometer (Specord 210, model Analytikjena) set to 450 nm as the method described by 48 .
Fatty acid analysis of Artemia.Lipid extraction: Weigh 2-20 g of the sample into a 250 mL centrifuge bottle; add sufficient water to bring total water present to 16 mL together with 40 mL methanol and 20 mL chloroform.Macerate for 2 min; add further 20 mL chloroform and macerate for 30 s; add 20 mL water and macerate again for 30 s. Centrifuge the mixture for 10 min at 2000-2500 rpm.Draw off the lower chloroform layer and filter through a coarse filter paper into a dry weighed flask or beaker.Evaporate the chloroform to dryness 49 .Methylation of lipid in a tube weighs 50 mg of lipid, add 5 mL of methanolic sulphuric acid (1 mL conc.sulphuric acid and 100 mL methanol) and 2 mL of benzene, close the tube well, and place in a water bath at 90 °C for an hour and a half.Cool, add 8 mL water and 5 mL petroleum ether, shake strongly, separate the ethereal layer in a dry tube and evaporate to dryness 50 .Coupling of two-dimensional thin layers chromatography with gas chromatography (GC) were used for the quantitative analysis of lipid classes and their constituent fatty acids.

Goldfish studied
Water quality analyses.Water quality parameters were measured twice per week in the midpoint of each aquarium.Temperature, pH, dissolved oxygen (DO), electric conductivity (EC) and total dissolved salts (TDS) were analyzed according to established protocols (APHA 1976) using Multi parameters probe meter (HI9829-03042-HANNA®insrruments, www.hanna inst.com).Total ammonia nitrogen (TAN) was estimated using a portable photometer (Martini MI 405 MR).According to 51 unionized ammonia (UIA) was determined from the preestimated TAN, temperature, and pH.
Growth performance and condition factor of goldfish.For each experimental treatment, 30 fish were randomly selected bi-weekly to estimate the periodical gain in weight and length.At the end of 90 days experimental period, goldfish were anesthetized by clove oil (Merck, Germany, 50 μL L −1 ), collected, counted, and weighed.The growth performance and feed utilization parameters were determined as follows: Proximate chemical composition of goldfish.Moisture, crude protein, crude lipid, fiber, and ash composition of experimental goldfish at the end of the experiment were determined using the analytical methodologies described in 47 .Triplicate samples (10 g dry matter/sample) from each experimental treatment were used for each analysis.
Analysis of β-carotene in experimental goldfish using high-performance liquid chromatography (HPLC).The triplicate sample (10 g) was used for detection of β-carotene in experimental goldfish using high-performance liquid chromatography (HPLC) according to 52 .The wavelength of detection was 476 nm.
Chromatic deformity and color intensity using computer-assisted image analysis.ImageJ 3 software was used to perform computer-assisted image analysis on photographs (National Institute of Mental Health, Bethesda, Maryland, USA).On the final day of the experiment, 15 randomly selected goldfish per each group (5 fish/replicate) were photographed under standard light conditions (lamp type: Philips TL-D Special 90; two tubes of F95, color temperature: 5300°K; one tube of F33, color temperature: 4100°K) using a digital camera (Olympus C-2000 Zoom; Olympus Optical, Tokyo, Japan).According to the procedures of 53 , grayscale values (0-255) of the R, G, and B components of the RGB scale were measured within a 30×30-pixel area of the red dorsal body surface.
Blood sampling and serum separation.The blood samples (six fish/replicate) were collected from the caudal vertebral vein of an anesthetized fish using a syringe (Masco Mid, Tanta city, Egypt) with finer gauge needle to Weight gain g fish −1 , WG = Wt − W0; Average daily gain ADG g fish −1 day −1 = Wt − W0 days −1 ; Specific growth rate % day −1 ; SGR = 100 × ln Wt − ln W0/experimental period , where: W0: initial fish weight g , Wt: final fish weight g , ln : natural logarithm; Survival rate (%) = 100 × (final number of fish/initial number of fish).
Vol www.nature.com/scientificreports/reduce the pain and for easy blood collection according to 54 at the end of the feeding experiment.The blood collected from each two fish was pooled to form one sample.The collected blood sample was divided into two tubes, one containing EDTA as an anticoagulant agent for hematological analysis.The other tube was without anticoagulant for serum separation.The blood was stored in a refrigerator for 4 h till colt formation then a centrifugation of blood clot was done at 4000 rpm/15 min at 4 °C and stored at − 20 °C until used for further analysis.Hematological analyses: The erythrocytes and leukocytes were counted according to the method described by 55 using neubauer hemocytometer and Natt-Herrik solution.Hemoglobin concentration was determined according to 55 using the cyanomet hemoglobin method Drabkin's solution.According to 56 , the microhematocrit method was conducted to determine the packed cell volume (PCV).In order to determine differential leukocytic count (DLC), thin blood films were prepared, air-dried, fixed with methanol for 3-5 min, stained with Gimsa stain for 8-10 min, and then allowed to dry.The white blood cells were counted among one hundred blood smears according to 55,57 .
Digestive enzymes activity: Lipase activity was determined colorimetrically at the wavelength of 580 nm 65 using commercial kits (REF:281 001 Spectrum, Egyptian company for Biotechnology, Egypt).Amylase activity was determined colorimetrically at the wavelength of 660 nm as the method described by 66 using commercial kits (CAT.NO.AY 10 50, Biodiagnostic Co. Egypt).
Immunological analysis: The lysozyme activity was assayed by ELISA according to the method described by 67 based on the ability of lysozyme to lyse Gram-positive lysozyme sensitive bacterium; Micrococcus lysodeikticus at the wavelength of 450 nm using the microplate ELISA reader.Immunoglobulin M (IgM) was assessed with an ELISA kit (Inova Biotechnology, China) 68 .Serum cortisol was determined by fish cortisol ELISA kit (Cusabio Biotech Co., LTD, China) 4 .
Histomorphological examination of liver and intestine.Liver and the whole intestine tissues of fish from different groups were used for histomorphological examination.The intestinal tissues (anterior, middle and posterior part) and liver samples (six samples/group) were collected and fixed in 10% formalin for 3 days.Then, the samples were dehydrated and washed multiple times in absolute alcohol before being embedded in paraffin.Serial 5-m longitudinal slices were cut on a Leica Rotary Microtome (RM 2145, Leica Microsystems) and placed on glass slides.The slides were then regularly stained with hematoxylin and eosin (H&E) 69 .The histomorphometric analysis of the intestine was performed using ImageJ analysis software (National Institutes of Health, MD, USA), whereas the intestinal villi length, width, and the inter-villi space were measured by ImageJ analysis software and expressed as µm.
Total RNA extraction, cDNA synthesis, and real-time quantitative PCR assay.Liver and brain samples (six samples/group) were collected in 2-mL sterile Eppendorf tubes from all tested groups.Then, the samples were immediately shocked in a liquid nitrogen.According to the manufacturer's instructions, total RNA was extracted from 50 mg of tissue using genazole (iNtRON Biotechnology).The integrity of extracted RNA was confirmed by ethidium bromide-stained 2% agarose gel electrophoresis.NanoDrop® BioDrop Spectrophotometer assessed RNA concentration.Following the manufacturer's instructions, 5 μg of extracted RNA sample was reverse transcribed using EasyScript First-strand cDNA synthesis Supermix (Oligo dT primer) (TransGenBiotechnology, China).Gene expression analysis was performed in the Rotor-Gene Q Real-time PCR system 70 using the TOPreal qPCR 2 × PreMIX (enzynomics, Korea) using goldfish gene-specific primers (Table 1) with β-actin as a normalizer (housekeeping) gene.The results were analyzed by Rotor-Gene Q series software 2.3.3;relative expression of selected genes was calculated by 2 −ΔΔCT method according to 71 .

Statistical analysis
After data validation for normality and homogeneity, statistical analysis was performed using GraphPad Prism 6.01 software.The data was analyzed using one-way analysis of variance (ANOVA) and Tukey's post hoc test to find out any significant differences between the tested groups when the P-value < 0.05.Means and standard error of the mean (SEM) were used to express the findings.

Study approval
The study is reported in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Proximate composition and characteristics of Artemia after 24 h-enrichment process
Table 2 displays the performance of Artemia after 24 h of enrichment, including survival (%), individual length (µm), total Artemia biomass (g L −1 ), and population density (numbers L −1 ).There were significant (P < 0.05) differences in the survival rate and biomass among Artemia groups.Low survival was recorded in SEA compared to the other Artemia groups.However, the largest total Artemia biomass production (g L −1 ) was recorded in SEA group.Moreover, there was a significant (P < 0.05) difference among experimental groups in population density and the lowest record was presented in the SEA.Non-significant (P > 0.05) differences were recorded in individual length of Artemia (µm) among groups.
The proximate chemical composition of the experimental Artemia (UEA, SEA, CEA, and SCA) is presented in Table 3.The SCA exhibited the highest levels of protein and total energy, and the lowest moisture content compared to the other groups of Artemia.While, SEA had the highest lipid content and fiber content compared to the other groups of Artemia.

Total carotenoids of Artemia
Figure 2 illustrates the total carotenoid content of un-enriched and enriched Artemia with spirulina and/or canthaxanthin used for feeding goldfish.Data showed significant differences (P < 0.05) among Artemia groups with the highest carotene content being detected in Artemia enriched with spirulina and canthaxanthin (SCA) followed by CEA and SEA.However, the lowest carotenoid content was detected in the UEA.

Goldfish
Water quality parameters Water quality parameters of goldfish aquariums are illustrated in Table 5.There were no significant (P > 0.05) differences among all treatments in terms of pH, temperature, dissolved oxygen (DO), total ammonia nitrogen (TAN), unionized ammonia (UIA), electric conductivity (EC), and total dissolved salts (TDS).

Growth performance and condition factor
Results of growth performance are illustrated in Table 6.There were significant (P < 0.05) differences among treatments in terms of final weight, weight gain, average daily gain (ADG), specific growth rate (SGR), final length, length gain and condition factor (K). Fish fed spirulina and canthaxanthin-enriched Artemia (T5) showed the highest values of growth performance, and lowest values of condition factor.3 represents the mean β-carotene concentrations (mg g −1 ) of goldfish fed commercial diet, unenriched Artemia, and Artemia enriched with spirulina and/or canthaxanthin.There was a significant (P < 0.05) difference between the experimental treatments in the concentration of β-carotene.The goldfish fed Artemia-supplemented diets exhibited greater (P < 0.05) β-carotene concentrations than control (T1) and unenriched Artemia group (T2).The highest (P < 0.05) β-carotene concentration was reported in T5 followed by T4 and T3 respectively.

Chromatic deformity and color intensity using computer-assisted image analysis
The assessment of chromatic deformities and color intensity in goldfish fed different feeding regimes are presented in Table 8.Significant differences (P < 0.05) in chromatic deformity among experimental treatments were detected.Goldfish fed commercial diet and spirulina and canthaxanthin-enriched Artemia (T5) had the lowest degree of chromatic deformities and a high level of color intensity and red index color.There were no significant differences (P > 0.05) between goldfish fed spirulina-enriched Artemia (T3) and canthaxanthin-enriched Artemia (T4).

Hematological parameters
Table 9 displays the haematological indices, including RBCs, Hb, PCV, WBCs, heterophil, lymphocytes and monocyte.There was an increase (P < 0.05) in RBCs, Hb, WBCs, and lymphocyte in goldfish fed the Artemia enriched diets (T3-T4-T5) compared to T2 and T1 with the highest values being reported in T5.    10 illustrates the serum biochemical analysis.The values of ALT, AST, cholesterol, triglycerides and glucose showed non-significant (P > 0.05) differences amongst experimental treatments.There were however, significant (P < 0.05) differences between experimental treatments in total protein, albumin and globulin with the highest values being observed in T5 and T4.The digestive enzyme activity (amylase, and lipase) presented in Fig. 4 showed no significant (P > 0.05) differences between experimental treatments.

Immune response parameters
Figure 5 displays the immune response [Lysozyme activity, immunoglobulin M, (IgM) and cortisol] of the experimental goldfish fed un-enriched and enriched Artemia with spirulina and/or canthaxanthin.There were variations (P < 0.05) in lysozyme activity and IgM levels between experimental treatments, with the highest values being observed in T5.However, there was no significant (P > 0.05) difference between experimental treatments regarding levels of cortisol.

Histomorphology of the intestine and liver
The morphometric analysis of intestinal tissues showed significant (P < 0.05) differences between experimental treatments (Table 11 and Fig. 6) in the different sections of intestine (anterior, middle and posterior).Villi length and goblet cells number in the anterior and middle portions of intestine and villi width and goblet cells number in the posterior portion of intestine showed significant (P < 0.05) differences between experimental treatments with the highest values being reported in T5.The histological investigations of liver tissue showed a similar anatomical arrangement with normal structure in the experimental treatments.Hepatocytes appeared polyhedral in shape, with nuclei usually spherical, big, and centered in the cell.The nucleolus was visible, and there were granules in the cytoplasm.The liver parenchyma, the cells were unevenly dispersed and divided by sinusoidal capillaries.Small blood arteries, bile ducts, and an exocrine pancreas with serous acinar cells with eosinophilic staining at the apical region and basophilic staining towards the base were also seen (Fig. 7).www.nature.com/scientificreports/Expression of growth hormone, somatolactin-β and somatolactin-α genes Fold changes of growth hormone, somatolactin β and somatolactin-α genes in the goldfish brain and liver fed un-enriched and enriched Artemia with spirulina and/or canthaxanthin were demonstrated in Fig. 8.There were variations (P < 0.05) in SL-α and GH gene expression among experimental treatments.In goldfish fed Artemia, there was an increase in GH gene expression in the liver and brain, with the greatest values being observed in T5.However, a decrease in SL-β gene expression was observed in the liver and brain (T5 < T4 < T3 < T2 < T1).SL-α gene expression was detected in the brain with higher expression in Artemia-enriched treatments, particularly in T5.

Discussion
The enrichment of brine shrimp, particularly during larval rearing, makes Artemia crucial to manage goldfish hatcheries.The main objective of Artemia enrichment is to improve Artemia nutritional value that has a significant effect on the quality of the fish larvae.
In the present study, the performance of Artemia after 24 h enrichment process revealed that the use of canthaxanthin powder improved the survival, and population density of canthaxanthin-enriched Artemia (CEA).However, spirulina improved total Artemia biomass of spirulina-enriched Artemia (SEA).Similar results were Table 11.Intestinal morphometric analysis (means ± SEM) of goldfish fed un-enriched and enriched Artemia with spirulina and/or canthaxanthin for 90 days.T1 = goldfish fed commercial diet; T2 = goldfish fed commercial diet and un-enriched Artemia; T3 = goldfish fed commercial diet and spirulina-enriched Artemia; T4 = goldfish fed commercial diet and canthaxanthin-enriched Artemia; T5 = goldfish fed commercial diet and spirulina and canthaxanthin-enriched Artemia.Means within the same row lack common superscripts are significantly different (P < 0.05).

Intestinal portion Variable
Experimental treatment  reported by 72,73 .Moreover, Ref. 74 found that feeding Artima on high β-carotene algae improved their performance.This may be due to Artemia biomass output being greatly influenced by the quantity and quality of food available, as well as the rising environment 75 .Moreover, canthaxanthin powder, used to enrich Artemia as an artificial source of carotenoids, can be easily dissolved in water with a smaller particle size than spirulina 72,76 .This may provide Artemia with its nutritional requirements.As a result, it is recommended to use canthaxanthin to improve Artemia survival rate and population density however; spirulina can be used to obtain higher biomass.Therefore, a mixture of canthaxanthin and spirulina appears to be a better choice in enriching brine shrimp.This study examined the proximate chemical composition of un-enriched Artemia (UEA), SEA, CEA, and SCA.It was found that, SEA, CEA, and SCA improved the chemical composition of Artemia in the form of higher protein, fat and total energy content, lower moisture, fiber and ash compared to UEA.SCA had a high protein and energy content, although SEA had a high-fat level.This result is in line with the findings of 77 .This may be attributed to spirulina's high fat and protein levels 78 and canthaxanthin's ability to motivate Artemia due to its high β-carotene content 79 .Therefore, canthaxanthin and/or spirulina can improve the chemical composition of brine shrimp.

P-value
In the current study, SCA had greater total β-carotene content than the other treatments.This may be due to the considerable pigmentation present in canthaxanthin and spirulina, which is naturally low in Artemia.The findings of this study agree with those reported by 74 who stated that carotenoids, which are originally stored in algal cells, can be fed to Artemia and can have a great impact on its health and performance.
According to the current study, SCA treatment had a high concentration of total polyunsaturated fatty acids (ƩPUFA), total unsaturated fatty acids (ƩUFA), total omega 3 (ƩOmega 3), and Eicosapentaenoic acids (EPA), and a very low concentration of total saturated fatty acids (ƩSFA), and total high unsaturated fatty acids (ƩHUFA).Numerous studies have also demonstrated that Artemia is a rich source of polyunsaturated fatty acids (PUFA), which are necessary for the growth and metabolism of many aquatic species 80,81 .Furthermore, PUFA, such as Eicosapentaenoic acids (EPA) and Docosahexaenoic acids (DHA), have been shown to have a significant impact on the early growth development and survival rate of goldfish larvae 82,83 .Thus, canthaxanthin and/ or spirulina can be effectively used as potent enrichment agents for improving Artemia growth performance, chemical composition, β-carotene content and polyunsaturated fatty acids concentration.
Maintaining optimal water conditions in a goldfish tank is crucial for effective keeping of goldfish in a tank and for improving its performance, especially during the larval rearing period, which is characterized by intense feeding with live-rich diets high in lipids and proteins.The water quality results of the current study revealed absence of any negative impacts of canthaxanthin and/or spirulina on goldfish water parameters with recorded values falling within the recommended ranges of goldfish.Similar water quality records were reported by 4 .This finding is compatible with that of 84 , who said that goldfish can be utilized to help maintain water quality in rearing tanks by eating algae and other residues such as lost feed.Goldfish can also be utilized to keep rearing tanks clean for longer periods of time 85 .
Goldfish growth rates rely entirely on the environment in which they are kept, as well as the amount of highquality food they are fed.In the current study, feeding enriched Artemia to goldfish increased their growth rate, particularly in the T5 treatment.The findings of this study are consistent with those of other studies indicating that feeding goldfish natural live food resulted in better growth than feeding artificial feed 3,79 .Moreover, Ref. 86 found that Artemia nauplii cannot meet the nutritional requirements for fish larval development when used as the only source of food due to the lack of essential polyunsaturated fatty acids.
The growth performance of goldfish was boosted by feeding them Artemia enriched with carotenoids, particularly Artemia supplemented with spirulina and canthaxanthin (T5) because of the high nutrition value of carotenoids, polyunsaturated fatty acids, unsaturated fatty acids (UFA), DHA, and omega3 (ω3) fatty acids present in spirulina and canthaxanthin-enriched Artemia (SCA) reported in the present study.Adult goldfish  www.nature.com/scientificreports/can grow on vegetable diets, however juvenile goldfish require a greater protein content in their feeds for better growth 87 .Furthermore, improved growth performance may be attributed to feeding goldfish live natural food rather than commercial diets which are dependent on the palatability, quality, and adequate quantity of feed, size, type, and physical appearance of the feed, as well as other factors such as attractiveness, feed acceptability, and assimilation of nutrients 3 .In the same context, Ref. 88 reported the greatest length growth of goldfish larvae fed Artemia (15.8 mm), followed by those fed Artemia plus a commercial feed (14.8 mm) and those just fed a commercial feed (10.8 mm length).In contrast, there is debate over the contribution of carotenoids to fish growth promotion.There was no discernible improvement in P. leopardus growth performance following treatment with varying astaxanthin concentrations 89 .Earlier studies found that fish given just a dry commercial feed had a poorer survival rate (61.9%) than those fed just Artemia (96.4%) or Artemia plus a commercial feed (94%).Reference 44 stated that the growth and survival of goldfish larvae were better demonstrated by the biofloc culture method than by the clear water culture system.Regardless of the cultivation system, the higher supply level of Artemia nauplii (300 Artemia nauplii/fish larvae) resulted in the best growth of the goldfish larvae as compared to the lower densities of nauplii.Therefore, canthaxanthin and/or spirulina can be effectively used as powerful Artemia enrichment agents for improving goldfish growth performance and survival.
After 90 days of feeding, the chemical composition of goldfish indicated an increase in the protein content in response to feeding enriched Artemia.This may be due to the relatively higher amount of protein in enriched Artemia compared to either un-enriched or commercial feed as presented in this study.This finding is similar to that reported by 90 .
Due to their inability to biosynthesize carotenoids properly, ornamental fish, like other animals, require proper nutrition for the development of natural pigmentation and good health 91 .Fish with higher carotenoid contents and fewer chromatic deformities typically have more color improvement.In the current study, goldfish fed spirulina and canthaxanthin-enriched Artemia (T5) had the lowest value of chromatic deformity.Same patterns were observed by 92,93 .The distinct colors of goldfish compared to other fish may help to explain this.Red, green, and blue color intensity levels (Red-Green-Blue; RGB) seem to have a role in how redness becomes visible in goldfish.The unweighted color intensity and R values are enhanced in T5.The starting concentration of redness and genetic background are believed to control the rate at which the red color intensity changes, which might explain the observed variations in red color intensity among fish fed live food from various treatments.The results of the current investigation are consistent with those of 53,94 .Moreover, Ref. 95 reported that feeding Artemia franciscana with Dunaliella salina rich in β-carotene improved platy fish (Xiphophorus maculatus) skin pigmentation and the skin color red index.
The increase in β-carotenoid content observed in this study when goldfish were fed spirulina and canthaxanthin-enriched Artemia (T5) as an exogenous source of carotenoid for three months was due to the fact that spirulina and canthaxanthin are both coloring agents [96][97][98] .Also, Ref. 95 discovered that Artemia franciscana supplemented with β-carotene from one type of algae called Dunaliella salina increased the skin pigmentation, red index of the skin color, and total carotenoid concentration in platy fish.Similarly, the levels of carotenoids in the skin of Astronotus ocellatus that were given Dunaliella salina, a natural source of β-carotene, were substantially greater in comparison to those who were given commercial feed, as stated by 79 , who added that the accumulation of carotenoids in the skin stimulates fish color modulations.Therefore, feeding goldfish spirulina and/or canthaxanthin-enriched Artemia improved red index and pigmentation of the skin.
Several studies on the hematological and immunostimulant benefits of carotenoids on ornamental fish have been conducted 74,79,99 .The hematological parameters of goldfish fed spirulina and canthaxanthin-supplemented Artemia (T5) were enhanced.The goldfish's hematological levels in this study were within normal ranges 100,101 .All Artemia-enriched treatments are lower in ALT and AST levels compared to T1.This result is comparable to that of 102 .Reduced ALT and AST levels have been frequently associated with great improvements in fish health, particularly the liver 103 .In the current study, goldfish fed spirulina-canthaxanthin enriched-Artemia (T5) had a higher total protein, albumin, total immunoglobulin, and lysozyme activity compared to fish fed commercial diet (T1).The current study findings are consistent with the findings of 99 .Albumin and globulins are two types of serum proteins.
The liver produces albumin and an osmotic force that keeps the circulatory space at a constant fluid volume.Albumin serves as a protein transporter and a protein reserve 104 .Lysozyme is a component of a fish defense system that causes bacterial lysis as well as activation of the complement system and phagocytes 105 .Feeding goldfish on spirulina and canthaxanthin-supplemented Artemia (T5) induced a considerable increase in lysozyme activity and immunoglobulin M (IgM) levels in the present study.This is consistent with the finding of 106 , who found that while lysozyme activity in the 100, 200, and 400 mg kg −1 mixed herbal enriched meal groups did not differ significantly from the control group during the first week, it did during the second and fourth weeks.Immunoglobulins mediate adaptive humoral immunity in fish, with IgM form the bulk of immune responses in teleost fish 107 .In addition, Ref. 89 concluded that dietary astaxanthin improved the antioxidant capacity, immunity and disease resistance of coral trout.
Carotene serves as a precursor to vitamin A, which is essential for immune system function, reduces free radicals, prevents lipid oxidation, preserves cell membrane flexibility, and reduces immunosuppressive peroxides Refs. 99,108found that β-carotene-enriched Artemia fed to fish improved non-specific immune responses in form of high total immunoglobulin, and lysozyme activity in comparison to the control group.Conclusively, feeding Artemia enriched with canthaxanthin and/or spirulina enhances the health status and immune response of goldfish.
The height and width of intestinal villi, as well as the number of goblet cells, directly affect the digestion and absorption of nutrients in fish 109 .In this study, histomorphometric examination of intestinal villi absorptive capacity revealed that T5 treatment showed an increase in villi length, villi width, and the number of goblet cells.The improvement in intestinal morphometry (height and width of intestinal villi, the number of goblet cells) www.nature.com/scientificreports/correlates with our findings of increased goldfish growth performance.The improvement in the nutritional or environmental conditions of the fish results in an improvement in the digestive system, which is reflected in the growth improvement.In this regard, Ref. 110 discovered that the Nile tilapia (Oreochromis niloticus) given Quillaja saponaria and/or Yucca schidigera had improved intestinal histomorphology (increase in intestinal villi length and width, decrease in the inter-villi gap, and increase in goblet cell count) compared to the control.A histological examination of the liver of goldfish fed only a commercial diet (T1) revealed that the hepatocytes were properly arranged when compared to those of other fish species.While feeding goldfish on spirulina and canthaxanthin-supplemented Artemia (T5) resulted in minimal vacuolar changes and significant glycogen accumulation and feeding goldfish canthaxanthin-supplemented Artemia (T4) resulted in mild vacuolar changes.This finding is consistent with the results of ALT and AST of goldfish reported also in the present study.Similar results were reported by 111,112 .Thus, using of Artemia enriched with canthaxanthin and/or spirulina as a source of goldfish nutrition improved intestine and liver health and therefore enhanced feed absorption capacity and growth performance.
Goldfish are commonly used as model fish in research on hormones, diet, behavior, and stress 113 .In the pars intermedia of the pituitary gland, somatolactin (SL), a member of the GH family, is produced and related to lipid metabolism 114 .Additionally, by aggregating melanosomes, SL how teleosts' bodies are colored 115 .Compared to the T1, GH expression was elevated in the liver and brain of fish in all treatments of the current experiment.There is no SL-α expression in the livers of goldfish because SL-α expression is not detected elsewhere other than the brain 116 .SL-β expression is suppressed in both the brain and liver.However, SL-α is raised in the brain, as shown by 117 , who stated that SL-α and proopiomelanocortin (POMC), as well as SL-β and vertebrate melanin-concentrating hormone (Pmch1), are responsible for the darkening and lightening of body color in response to the background color.Members of the growth hormone (GH) family and somatolactin (SL) modulate energy metabolism, are also associated with teleost body color modulation because these hormones are involved in both the control of body color and energy metabolism.Reference 117 investigated the effects of feeding status and background color on the expression of genes involved in the control of body color in goldfish and discovered that feeding status may influence the modulation of body color via SL-α and SL-β, however, these effects may be restricted in comparison to the effect of background color.

Conclusion
The use of canthaxanthin and/or spirulina enhanced the protein percentage, β-carotene content, survival rate, and performance of Artemia.Goldfish that were fed enriched Artemia had improved immune-physiological, transcriptome, and pigmentation responses in addition to improved performance.When goldfish were fed on Artemia that was enhanced with both spirulina and canthaxanthin, the best profile of improved health and performance was achieved.

Figure 3 .
Figure 3.Mean concentrations of β-carotene (mg g −1 ) of experimental goldfish extracted and measured using high-performance liquid chromatography (HPLC).Means within the same column lack common superscripts are significantly different (P < 0.05).T1 = goldfish fed commercial diet; T2 = goldfish fed commercial diet and un-enriched Artemia; T3 = goldfish fed commercial diet and spirulina-enriched Artemia; T4 = goldfish fed commercial diet and canthaxanthin-enriched Artemia; T5 = goldfish fed commercial diet and spirulina and canthaxanthin-enriched Artemia.

Figure 4 .
Figure 4. Means ± SEM of digestive enzyme activity [Amylase activity (A) and Lipase activity (B)] of experimental goldfish fed un-enriched and enriched Artemia with spirulina and/or canthaxanthin for 90 days.Means within the same column lack common superscripts are significantly different (P < 0.05).T1 = goldfish fed commercial diet only; T2 = goldfish fed commercial diet and un-enriched Artemia; T3 = goldfish fed commercial diet and spirulina-enriched Artemia; T4 = goldfish fed commercial diet and canthaxanthin-enriched Artemia; and T5 = goldfish fed commercial diet and spirulina and canthaxanthin-enriched Artemia.

Figure 5 .
Figure 5. Means ± SEM of immune response [Lysozyme activity (A), immunoglobulin M, IgM (B) and cortisol (C)] of experimental goldfish fed un-enriched and enriched Artemia with spirulina and/or canthaxanthin for 90 days.Means within the column lack common superscripts are significantly different (P < 0.05).T1 = goldfish fed commercial diet; T2 = goldfish fed commercial diet and un-enriched Artemia; T3 = goldfish fed commercial diet and spirulina-enriched Artemia; T4 = goldfish fed commercial diet and canthaxanthin-enriched Artemia; T5 = goldfish fed commercial diet and spirulina and canthaxanthin-enriched Artemia.

Table 4 .
Fatty acid profile (% of total fatty acids) of Artemia used for feeding experimental goldfish.

Table 5 .
Table 7illustrates the approximate chemical composition of goldfish fed unenriched and enriched Artemia with spirulina and/or canthaxanthin.Dry matter, moisture, crude protein, crude fat, fiber, ash, carbohydrate, and energy varied significantly (P < 0.05) among experimental goldfish.The goldfish fed spirulina and canthaxanthinenriched Artemia (T5) had the highest protein levels, however the goldfish fed commercial diet and spirulinaenriched Artemia (T3) group had the highest carcass energy levels.Moreover, the highest crude fat level was reported in T2 and T4, and the highest carbohydrate level was recorded in T1.Water quality parameters (means ± SEM) of goldfish fed un-enriched and enriched Artemia with spirulina and/or canthaxanthin for 90 days.T1 = goldfish fed commercial diet; T2 = goldfish fed commercial diet and un-enriched Artemia; T3 = goldfish fed commercial diet and spirulina-enriched Artemia; T4 = goldfish fed commercial diet and canthaxanthin-enriched Artemia; T5 = goldfish fed commercial diet and spirulina and canthaxanthin-enriched Artemia.DO Dissolved oxygen, TAN total ammonia nitrogen, UIA unionized ammonia, EC electric conductivity, TDS total dissolved salts.

Table 7 .
Proximate chemical composition on dry matter basis (means ± SEM) of goldfish fed un-enriched and enriched Artemia with spirulina and/or canthaxanthin for 90 days.T1 = goldfish fed commercial diet; T2 = goldfish fed commercial diet and un-enriched Artemia; T3 = goldfish fed commercial diet and spirulinaenriched Artemia; T4 = goldfish fed commercial diet and canthaxanthin-enriched Artemia; T5 = goldfish fed commercial diet and spirulina and canthaxanthin-enriched Artemia.Means within the same row lack common superscripts are significantly different (P < 0.05).

Table 8 .
The Means within the same row lack common superscripts are significantly different (P < 0.05).

Table 9 .
Hematological analysis (means ± SEM) of goldfish fed un-enriched and enriched Artemia with spirulina and/or canthaxanthin for 90 days.T1 = goldfish fed commercial diet; T2 = goldfish fed commercial diet and un-enriched Artemia; T3 = goldfish fed commercial diet and spirulina-enriched Artemia; T4 = goldfish fed commercial diet and canthaxanthin-enriched Artemia; T5 = goldfish fed commercial diet and spirulina and canthaxanthin-enriched Artemia.RBCs red blood cells, HB hemoglobin, PCV packed cell volume, WBCs white blood cells.Means within the same row lack common superscripts are significantly different (P < 0.05).